Distribution characteristics of the Legionella CRISPR-Cas system and its regulatory mechanism underpinning phenotypic function.

Legionella is a common intracellular parasitic bacterium that infects humans via the respiratory tract, causing Legionnaires' disease, with fever and pneumonia as the main symptoms. The emergence of highly virulent and azithromycin-resistant Legionella pneumophila is a major challenge in clinical anti-infective therapy. The CRISPR-Cas acquired immune system provides immune defense against foreign nucleic acids and regulates strain biological functions. However, the distribution of the CRISPR-Cas system in Legionella and how it regulates gene expression in L. pneumophila remain unclear. Herein, we assessed 915 Legionella whole-genome sequences to determine the distribution characteristics of the CRISPR-Cas system and constructed gene deletion mutants to explore the regulation of the system based on growth ability in vitro, antibiotic sensitivity, and intracellular proliferation of L. pneumophila. The CRISPR-Cas system in Legionella was predominantly Type II-B and was mainly concentrated in the genome of L. pneumophila ST1 strains. The Type II-B CRISPR-Cas system showed no effect on the strain's growth ability in vitro but significantly reduced resistance to azithromycin and decreased proliferation ability due to regulation of the lpeAB efflux pump and the Dot/Icm type IV secretion system. Thus, the Type II-B CRISPR-Cas system plays a crucial role in regulating the virulence of L. pneumophila. This expands our understanding of drug resistance and pathogenicity in Legionella, provides a scientific basis for the prevention of Legionnaires' disease outbreaks and the rational use of clinical drugs, and facilitates effective treatment of Legionnaires' disease.

Retrospective Examination of Q Fever Endocarditis: An Underdiagnosed Disease in the Mainland of China.

BACKGROUND: Q fever endocarditis, a chronic illness caused by Coxiella burnetii, can be fatal if misdiagnosed or left untreated. Despite a relatively high positive rate of Q fever serology in healthy individuals in the mainland of China, very few cases of Q fever endocarditis have been reported. This study summarized cases of Q fever endocarditis among blood culture negative endocarditis (BCNE) patients and discussed factors attributing to the low diagnostic rate. METHODS: We identified confirmed cases of Q fever endocarditis among 637 consecutive patients with infective endocarditis (IE) in the Peking Union Medical College Hospital between 2006 and 2016. The clinical findings for each confirmed case were recorded. BCNE patients were also examined and each BCNE patient's Q fever risk factors were identified. The risk factors and presence of Q fever serologic testing between BCNE patients suspected and unsuspected of Q fever were compared using the Chi-squared or Chi-squared with Yates' correction for continuity. RESULTS: Among the IE patients examined, there were 147 BCNE patients, of whom only 11 patients (7.5%) were suspected of Q fever and undergone serological testing for C. burnetii. Six out of 11 suspected cases were diagnosed as Q fever endocarditis. For the remaining136 BCNE patients, none of them was suspected of Q fever nor underwent relevant testing. Risk factors for Q fever endocarditis were comparable between suspected and unsuspected patients, with the most common risk factors being valvulopathy in both groups. However, significantly more patients had consulted the Infectious Diseases Division and undergone comprehensive diagnostic tests in the suspected group than the unsuspected group (100% vs. 63%, P= 0.03). CONCLUSIONS: Q fever endocarditis is a serious yet treatable condition. Lacking awareness of the disease may prevent BCNE patients from being identified, despite having Q fever risk factors. Increasing awareness and guideline adherence are crucial in avoiding misdiagnosing and missed diagnosing of the disease.

Molecular detection of spotted fever group Rickettsia in Dermacentor silvarum from a forest area of northeastern China.

In total, 676 Dermacentor silvarum Olenev (Acari: Ixodidae) from a forest area of Jilin Province in northeastern China were examined by polymerase chain reaction for the presence of spotted fever group (SFG) Rickettsia. The overall positive rate was 10.7%, with a 95% confidence interval from 8.3 to 13.0%. The SFG Rickettsia infection was more prevalent in adults than in nymphs, and in fed ticks obtained from domestic animals than in those collected on vegetation. Sequence analysis of the partial outer membrane protein A gene confirmed the existence of R. sibirica and discovered a novel rickettsial agent in this area, the sequence of which was identical to that of DnS14 genotype Rickettsia previously reported in the former Soviet Union.

[Development of a quantitative real-time polymerase chain reaction for detecting Bartonella henselae].

OBJECTIVE: To develop a quantitative real-time polymerase chain reaction (PCR) for detecting Bartonella henselae. METHODS: According to the 16S-23S rRNA intervening sequences (IVS) specific for B. henselae, one pair of primers and one TaqMan-MGB probe were designed. A quantitative real-time PCR was developed with the primers, the probe, and the IVS, a standard template, in DNA sequence detection system (ABI 7900HT). RESULTS: The standard curve was established with the standard template and the relationship between the value of threshold cycle (Ct) and the DNA copy number was linear (r = 0.997). The sensitivity of this quantitative real-time PCR was about 1000 times higher than that of a common PCR used to detect homologous DNA. By this quantitative real-time PCR, the DNA sample of B. henselae was positively detected but not from other rickettsial or bacterial DNA samples. The variation coefficients of intra- and inter-assay reproducibility were 0.2%-1.9%. Using the real-time quantitative PCR to detect samples from mice that were experimentally infected with B. henselae, the small amount of B. henselae DNA was detected in blood samples on days 2, 3, and 5 and large amount of B. henselae DNA was detected in spleen samples on days 1 and 2 after infection. CONCLUSION: Results from our study suggested that this quantitative real-time PCR was highly specific, sensitive and with good repeatability for detection of B. henselae. It seemed quite useful for rapid detection of tiny DNA of B. henselae in various samples and laboratory diagnosis of bartonellosis caused by B. henselae.

[Study on the development of a real-time quantitative polymerase chain reaction assay to detect Rickettsia].

OBJECTIVE: To develop a real-time quantitative polymerase chain reaction(PCR) assay for detecting Rickettsia rickettsii. METHODS: The primers and TaqMan-MGB probe were designed according to the ompB gene of R. rickettsii. A DNA fragment of ompB gene amplified from R. rickettsii by PCR was used as a standard template for the development of the method. RESULTS: 5 copies of ompB fragments of R. rickettsii were detected. The genomic DNA of R. rickettsii was detected by the developed quantitative PCR assay. However, the genomic DNA from another rickettsial or bacterial agent was not determined. Through this developed method, the positive results were obtained from the animals and cells, artificially infected with R. rickettsii. CONCLUSION: The real-time quantitative PCR assay seemed to be highly sensitive and specific which might be used to rapidly detect R. rickettsia DNA in various samples and to early diagnose patients infected by R. rickettsii.

[Detection of Rickettsia prowazekii by quantitative real-time PCR].

OBJECTIVE: To develop a quantitative real-time polymerase chain reaction (PCR) for detecting Rickettsia prowazekii. METHODS: Primers and TaqMan-MGB probes designed based on ompB gene of R. prowazekii, were used to develop this method. RESULTS: For the quantitative real-time PCR, the relationship between the values of threshold cycle (Ct) and the DNA copy number was linear (r = 0.999) and the sensitivity was about 100 times higher than that of the nested PCR for detecting the same DNA sample. The results of the genomic DNA samples of other rickettsial and bacterial agents detected by real-time PCR were all negative. DNAs extracted from blood samples of guinea pig infected with R. prowazekii were examined by real-time PCR and the positive results were obtained from some of these samples. However, the results of some samples in nested PCR assay were all negative. CONCLUSION: These results suggested that the real-time PCR was highly specific and sensitive for detection of R. prowazekii that was useful for the detection of tiny DNA of R. prowazekii in blood samples from patients suspected of having epidemic typhus.

Recombinant 56-kilodalton major outer membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity.

The gene encoding the 56-kDa protein of Orientia tsutsugamushi Shanxi was amplified by a nested PCR and cloned into the expression vector pQE30. The 56-kDa protein of O. tsutsugamushi Shanxi (Sxh56) was expressed as a fusion protein with the His(6)-binding protein of Escherichia coli by deleting the signal peptide-encoding sequence from the 5' end of the open reading frame. The recombinant protein formed inclusion bodies when expressed in E. coli M15. The recombinant protein was examined for reactivity with mouse sera against three antigenic prototypes of O. tsutsugamushi by an immunoblot assay. The recombinant Sxh56 reacted only to polyclonal antiserum to O. tsutsugamushi Gilliam in an enzyme-linked immunosorbent assay (ELISA) and in an immunoblot assay. Recombinant Sxh56 was purified by Ni-nitrilotriacetic acid affinity chromatography and injected into mice to evaluate its ability to stimulate immune responses. High levels of immunoglobulin G and T-cell proliferation appeared in mice immunized with the recombinant protein. The recombinant Sxh56 was used in an ELISA to evaluate the ability of the method to detect antibodies to O. tsutsugamushi in human and animal sera. Thirty sera from mice infected with O. tsutsugamushi Gilliam or Shanxi and 55 sera from normal mice were detected in the ELISA with recombinant Sxh56, and the sensitivity and specificity were 96.67 and 100%, respectively. One hundred fifty-one positive sera and 412 negative sera to O. tsutsugamushi Gilliam were detected in an indirect immunofluorescence assay with the recombinant protein, and the sensitivity and specificity were 96.36 and 88.08%, respectively. These results strongly suggest that the recombinant Sxh56 is a suitable type-specific immunodiagnostic antigen and vaccine candidate.

Prevalence of Anaplasma phagocytophila and Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China.

A total of 1,345 Ixodes persulcatus ticks collected from northeastern China were investigated for the presence of Anaplasma phagocytophila and Borrelia burgdorferi by a nested polymerase chain reaction (PCR). Sixty-two (4.6%) ticks were positive for A. phagocytophila and 454 (33.8%) were positive for B. burgdorferi. Seven (0.5%) were coinfected with both agents. Sequence analysis of 919-basepair PCR amplicons revealed three types of A. phagocytophila. Type 1 was identical to the published sequences of A. phagocytophilas responsible for human granulocytic ehrlichiosis (HGE). The other two variants differed from the HGE agent sequence at one and four positions, respectively. These findings imply that infection with A. phagocytophila poses a potential health threat to both humans and animals in northeastern China, and that ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite, particularly when clinical manifestations are atypical for Lyme disease.